Zou et al. BMC Psychiatry (2020) 20:388 https://doi.org/10.1186/s12888-020-02790-y

RESEARCH ARTICLE Open Access The association between serotonin-related polymorphisms and susceptibility and early sertraline response in patients with panic disorder Zhili Zou, Yulan Huang, Jinyu Wang, Wenjiao Min and Bo Zhou*

Abstract Background: A number of studies have shown that genetic factor plays an important role in etiology of panic disorder (PD). The aim of the present study was to examine the association of serotonin-related gene polymorphisms with PD risk. Then, we analyzed the correlation between these gene polymorphisms and response to sertraline drug. Methods: Two hundred thirty-three patients with PD and 231 healthy controls were enrolled in the study. Panic Disorder Severity Scale (PDSS) were administered to all subjects, and all patients in the study were also assessed after 4 weeks of treatment. The SLC6A4(rs140701, rs3813034, 5-HTTLPR and STin2), 5-HTR1A rs6295, 5-HTR2A rs6313 and COMT rs4680 gene polymorphisms were genotyped and assessed for the potential association. Results: The allelic model showed that the SLC6A4 rs140701 polymorphism variant was significantly associated with increased risk of PD (OR = 0.624, 95% CI 0.450–0.864, p < 0.05), and a significant result was found in the dominant model (OR = 0.546; 95% CI, 0.371–0.804, p < 0.05). There was a significant difference in allele and genotype frequency between responders and nonresponders in the 5-HTTLPR polymorphism (OR = 0.205, 95% CI 0.128–0.328; OR = 0.249, 95% CI 0.155–0.401, both p < 0.001), indicating the PD patients with S-allele had a poorer response to sertraline than L-allele carriers. Conclusions: The present study suggests that the SLC6A4 rs140701 polymorphism variant may be associated with susceptibility to PD, and 5-HTTLPR polymorphism may be a predictor of response to sertraline in the treatment of PD. Keywords: Panic disorder, Serotonin transporter, Polymorphism, Setraline

* Correspondence: [email protected] Psychosomatic Department, Sichuan Academy of Medical Science & Sichuan Provincial People’s Hospital, No. 32 West Second Section First Ring Road, Chengdu, Sichuan 610072, P.R. China

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zou et al. BMC Psychiatry (2020) 20:388 Page 2 of 9

Background events [33], was the first-line treatment for PD. Hence, the Panic disorder is a common anxiety disorder character- first aim of this study was to investigate the association of ized by sudden and unexpected panic attacks and ac- serotonin-related gene polymorphisms including SLC6A4 company with obviously anticipatory anxiety. The (rs140701, rs3813034, 5-HTTLPR and STin2), 5-HTR1A estimated lifetime prevalence of PD is 3.4 to 4.7% [1, 2]. rs6295, 5-HTR2A rs6313 and COMT rs4680 with PD risk, It typically occurs in young adults, and women are more and the second aim was to examine the association be- likely to be affected than men [3]. However, the etiology tween these single nucleotide polymorphisms (SNPs) and of PD is complex and unclear. A meta-analysis showed early response to sertraline treatment in PD. genetic factors explain approximately 43% of the vari- ance in the PD [4], indicating an important role in the Methods pathological PD. Previous studies have consistently dem- Participants onstrated serotonin involvement in the neurobiology of Two hundred thirty-three patients with PD were recruited PD [5, 6]. Clinical studies also demonstrated that select- from inpatient and outpatient populations in the Depart- ive serotonin-reuptake inhibitors (SSRIs) increasing the ment of Psychosomatic, Sichuan Provincial People’sHos- synaptic availability of 5-HT and are effective in the pital between May 2015 and December 2018(recruitment treatment of PD [7]. Therefore, the certain variants in flow chart in Fig. S1). Diagnosis of PD was conducted ac- the serotonin-related , such as serotonin trans- cording to the Structured Clinical Interview for the Diag- porter (5-HTT), 5-HT1A receptor (5-HTR1A), 5-HT2A nostic and Statistical Manual of Mental Disorders fourth receptor (5HTR2A) and catechol-O-methyltransferase edition (DSM-IV) (SCID-1) [34], which was administered (COMT) genes, may influence 5-HT neurotransmission, by trained clinical psychiatrists. Patients with neurological and they are good candidates for the study of PD. A few diseases, and/or past or current episodes of MDD, gener- studies have investigated the association between the 5- alized anxiety disorder(GAD), manic disorder, bipolar dis- HTT linked polymorphic region (5-HTTLPR), 5-HTR1A order, or any other psychiatric disorders rs6295, 5-HTR2A rs6313, COMT rs4680 polymorphisms were excluded. In addition, 231 healthy controls were re- and PD [8–16]. However, the results were hardly replic- cruited from the Center of Health Examination, Sichuan able and the pathogenesis of PD remains to be clarified. Provincial People’s Hospital. The SCID-1 was also per- It is important to note that previous studies have been formed to exclude a lifetime or current diagnosis of PD, conducted in different ethnic groups, few of them have MDD, GAD, among others. examined the relationship between these gene polymor- All subjects in this study were Han Chinese, and all phisms and PD in Chinese population. subjects were free of acute or chronic somatic disorders. SSRIs are recommended as the first-line antidepres- All patients were free of antidepressants or other psy- sants in treatment for PD. However, not all patients can chotropic medications intake within 2 weeks before their gain full response from these antidepressants. With the examination. Demographic data and clinical presenta- advancement of pharmacogenomic technologies, genetic tions were obtained from medical records or qualified variation has been identified to contribute to individual interviews. A 3-mL EDTA-anticoagulated peripheral response to antidepressants. Pharmacogenomic studies blood sample was collected from every individual. focusing on candidate gene polymorphisms implicated in antidepressant response, especially in the serotonergic Measures pathway. Abundant evidence has shown that serotonin- Panic disorder severity scale(PDSS) related gene polymorphisms played an important role in The Panic Disorder Severity Scale is a valid and reliable the clinical effects of antidepressants. For example, a few inventory. Seven items are included in this scale, and studies investigated that 5-HTTLPR, intron 2 (STin2), 5- each item is scored 0–4 on a Likert scale ranging from HTR1A rs6295, 5-HTR2A rs6313 and COMT rs4680 “none” to “extremely severe” [35]. Also, the scale was polymorphisms associated with antidepressant response translated into Chinese, and the PDSS-Chinese Ver- [17–26]. However, these findings are inconsistent and do sion(PDSS-CV) has good internal consistency (Cron- not completely explain the predictive effects of antidepres- bach’s alpha) with the overall score (0.83) [36]. sants. To our knowledge, many previous studies have been conducted in other psychiatric disorders, such as major Treatment depressive disorder(MDD) [27–30], and few of them have Setraline (100-200 mg qd) was administered to all pa- conducted in PD. In addition, the different results of these tients for 4-week period. Other psychotropic medica- studies might also be partly attributable to the differences tions were not permitted during the study except for in the type of antidepressant drug [31, 32]. Sertraline, a benzodiazepine, which was prescribed occasionally for SSRI which has been used widely to treat PD for its prom- insomnia with a minimal dosage at bedtime. PDSS was inent therapeutical effects and low incidence of adverse assessed to all patients at baseline and after 4 weeks of Zou et al. BMC Psychiatry (2020) 20:388 Page 3 of 9

treatment. Response was defined as a 40% or greater re- based on the distributions of allelic frequencies and gen- duction in scores on the pre-treatment PDSS [37]. etic models (additive, dominant, and recessive model), and odds ratios (ORs) and 95% confidence intervals (CIs) SNP selection and genotyping were performed in unconditional logistic regression ana- The SNP selected was based on literature search and lysis using PLINK v1.07, 20 adjusting for age, gender, minor allele frequency(MAF) > 0.05 of east Asian popula- educational level and resident location. To protect from tion from public databases. The references were listed in Type I error, a Bonferroni correction was conducted. the Table S1. SNP genotyping was performed using an im- For all analyses, statistical tests were two-tailed and an proved multiplex ligation detection reaction (iMLDR) alpha level of 0.05 was used to define statistical signifi- technique which developed by Genesky Biotechnologies cance. Power calculation (α = 0.05) was performed using Inc. (Shanghai, China). The primer information of the re- Power and Sample Size Calculation Software (3.6.1). action mixtures is described in Tables S2 and S3.The 20 μl multiplex polymerase chain reaction (PCR) included Results 1 x GC-I buffer (Takara), 0.3 mM dNTP, 3.0 mM Mg2+,1 Demographic data and clinical manifestations U HotStar Taq Polymerase (Qiagen Inc), 1 μlgenomic A total of 233 patients with PD (92 men, 141 women) DNA(5-10 ng/μl), and 1 μl Multiplex-PCR primermix. In and 231 controls (98 men, 133 women) were selected. addition, The 5-HTT of PCR reaction volume(10 μl) in- The average age of the study sample was (35.65 ± 9.77) cluded 10 x buffer I (Qiagen Inc), Q Solution(Qiagen years, and the mean age of the control group was Inc.), 0.2 mM Mg 2+, 1 U HotStarTaq polymerase (Qiagen (36.96 ± 7.82) years. 53.6% of the patients (n = 125) were Inc.), 1 μl genomic DNA, and 1 μl PCR primermix. The residing in urban locations, and 46.4% of the patients PCR cycling program was as follows: denaturation at 95 °C (n = 108) were residing in rural locations. No statistically for 2 min, followed by 11 cycles of 94 °C for 20s, annealing significant differences were noted between cases and at 65 °C for 40 s and 72 °C for 1.5 min, and each cycle de- controls in terms of sex, age, education level and resi- creased 0.5 °C. The third step was set as 24 cycles of 94 °C dent location(p > 0.05). In addition, the mean total dur- for 20s, 59 °C for 30 s, finally, 72 °C for 2 min and 4 °C for ation of panic disorder was (2.49 ± 1.45) years. The hold. In addition, the 5-HTT of cycling program for PCR PDSS score was 15.47 ± 3.82 before medication and was 95 °C for 2 min, 35 cycles of amplification consisted of 9.51 ± 3.81 after 4 weeks of medication, and the score 94 °C for 20 s and 72 °C for 1.5 min, and final extension at was significantly reduced after medication (p < 0.05). Ac- 68 °C for 60 min and a hold at 4 °C. 5 U SAP and 2 Exonu- cording to the definition of remission, the response rate cleaseIwere used to purify the PCR product at 37 °C and was 42.1%(n = 98). The demographic details of the sam- then 15 min of 75 °C for inactivation. 2 μlpurifiedmulti- ple are given in Table 1. plex PCR product, together with 6 μlddH2O, were added into the ligation reaction containing 1 μl of 10× ligation Association of serotonin-related gene polymorphisms buffer, 0.25 μl Tag DNA ligase, 5′ ligationprimer (1 μM) with PD risk 0.4 μl, 3′ ligationprimer (2 μM) 0.4 μl. The ligation cycling To determine whether the sample size of this study has program was set as 38 cycles× (94 °C for 1.5 min and 56 °C power to detect the real difference, a power analysis was for 4 min), and a hold at 4 °C. After PCR, 0.5 μlligation performed. From the result of Table S4, the sample size product was taken for Sequencing with 0.5 μl Liz500 Size of 233 vs 231 ensured that a two-sided test with α = 0.05 Standard, and 9 μl HiDi mixture (ABI3730XL). The raw had 80% power to detect an relative risk of 2.0 (Ψ = 2.0). data were analyzed by Gene Mapper v4.1 software All selected SNPs fulfilled the HWE in both cases and (AppliedBiosystems, USA). All primers, probes and label- controls(p > 0.05). To determine the extent of linkage ing oligos were designed by and ordered from Genesky disequilibrium (LD) between the two polymorphisms Biotechnologies Inc. (rs140701, rs3813034) in SLC6A4 gene, standardized LD coefficient D’ was calculated for this pair of polymor- Statistical analysis phisms. The R2 and D’ was 0.510 and 0.717 respectively, The data were analyzed using SPSS version 18.0 software indicating weak LD between these two polymorphisms. (SPSS Inc., Chicago, IL, USA). Student’s t-tests were The allele distributions of SLC6A4 (rs140701 and used for inter group comparisons of continuous vari- rs3813034) were significantly different between PD cases ables, with Pearson’s chi-square tests utilized for cat- and the controls (OR = 0.624, 95% CI 0.450–0.864, p = egorical variables. P values for the Hardy-Weinberg 0.004; OR = 0.705, 95% CI 0.509–0.975, p = 0.034; re- equilibrium (HWE) were tested by Pearson’s chi-square spectively). However, only SLC6A4 rs140701 remained test, and P > 0.05 indicated no significant deviation in al- significant after adjusting for Bonferroni’s multiple test- lele and genotype frequencies among subjects. Associa- ing(p = 0.028). After adjustment for age, gender, educa- tions between SNPs and disease status were determined tional level and resident location, the dominant model of Zou et al. BMC Psychiatry (2020) 20:388 Page 4 of 9

Table 1 Demographic and clinical characteristics in patients with PD and controls Variable PD (n = 233) Controls(n = 231) t/χ 2-value p-value Sex, n (%) Female 141(60.5) 133 (57.6) 0.414 0.520 Male 92 (39.5) 98 (42.4) Age 35.65 ± 9.77 36.96 ± 7.82 1.594 0.112 Educational level, n (%) < Junior high school 49 (21.0) 42 (18.2) High school 95 (40.8) 80 (34.6) 3.836 0.147 College and above 89 (38.2) 109 (47.2) Resident location, n (%) Urban 125 (53.6) 129 (55.8) Rural 108 (46.4) 102 (44.2) 0.266 0.635 Total duration of PD, years 2.49 ± 1.45 PDSS baseline 15.47 ± 3.82 PDSS 4-week 9.51 ± 3.81

SLC6A4 rs140701 showed the relative risk of PD for of the other gene polymorphisms were not significantly genotype CC + CT was lower than for genotype TT different between responders and nonresponders (p > (OR = 0.546; 95% CI, 0.371–0.804, p = 0.004), and after 0.05). (Table 3). Bonferroni correction for multiple comparisons, the sig- nificance remained (p = 0.028). In addition, the dominant model of rs3813034 showed Discussion the relative risk of PD for genotype CA + AA was lower In this present study, a significant relationship was found than for genotype CC (OR = 0.636; 95% CI, 0.432–0.935, between the SLC6A4 rs140701 polymorphism and PD. p = 0.025). For 5-HTTLPR polymorphism, the relative The patients with PD possessed significantly higher fre- risk of PD for genotype LL + LS was lower than for quencies of the TT genotype compared to the controls. genotype SS (OR = 0.632; 95% CI, 0.436–0.916; p = This result is consistent with the previous study reported 0.021). The additive model of rs140701 and rs3813034 that only SNP rs140701 was associated with PD in a 163 showed similar association with PD(OR = 0.638, 95% CI sample of African American [9]. Similar studies have 0.463–0.879, p = 0.009; OR = 0.721, 95% CI 0.526–0.988, found a nominally significant association of rs140701 p = 0.048; respectively). However, these correlations were with social anxiety disorder [38]. The 5-HTT is encoded no longer significant after adjusting for Bonferroni’s by the solute carrier family 6 member 4 (SLC6A4) gene, multiple testing (p > 0.05). Also, there were no signifi- at locus 17q11.2. SNP rs140701 is located in intron 9 of cant associations of other SNPs with PD in allelic or the serotonin transporter gene. SLC6A4 is involved in other models (p > 0.05) (Table 2). the transport of serotonin from synaptic spaces to pre- synaptic neurons, and the pool of available serotonin for Association between serotonin related gene subsequent release [39]. Collective evidence suggests the polymorphisms and early response to sertralines in the involvement of the serotonin system in the neurobiology treatment of PD and pharmacotherapy of PD [5–7]. Specific SLC6A4 var- Finally, we investigated whether variations of gene could iants may be associated with PD. In addition, previous predict response to sertraline in Han Chinese population studies have found 5-HTT knockout mice showed in- with PD. There was a significant difference in allele and creased anxiety-like behaviour. Such cases have been genotype frequency between responders and nonre- shown in an animal experiment which found homozy- sponders in the 5-HTTLPR polymorphism (OR = 0.205, gous 5-HTT knockout mice were more anxious [40, 41], 95% CI 0.128–0.328; OR = 0.249, 95% CI 0.155–0.401, all while 5-HTT overexpressing mice displayed reduced pcorr = 0.000; respectively), and the dominant and reces- anxiety-like behaviour [42]. These findings suggest that sive model of 5-HTTLPR showed significant association the importance of the SLC6A4 gene polymorphisms in with therapeutic response, indicating the PD patients genetic association studies of PD. However, the SNP with S-allele had a poorer response to sertraline than L- rs140701 of function is unclear and remains to be clari- allele carriers. However, genotype and allele frequencies fied in future studies. Zou et al. BMC Psychiatry (2020) 20:388 Page 5 of 9

Table 2 Association of serotonin-related gene polymorphisms with PD risk in Chinese Han population Gene SNP Alleles and Genotypes PD Controls Model OR (95% CI) p-value p-valuecorr (n, %) (n = 233) (n = 231) SLC6A4 rs140701 C 76 (16.3) 110 (23.8) Allelea 0.624 (0.450–0.864) 0.004 0.028 T 390 (83.7) 352 (76.2) CC 10 (4.3) 13 (5.6) Additiveb 0.638 (0.463–0.879) 0.009 0.063 CT 56 (24.0) 84 (36.4) Dominantb 0.546 (0.371–0.804) 0.004 0.028 TT 167 (71.7) 134 (58.0) Recessiveb 0.752 (0.323–1.751) 0.438 1 rs3813034 C 386 (82.8) 357 (77.3) Allelea 0.705 (0.509–0.975) 0.034 0.238 A 80 (17.2) 105 (22.7) CC 164 (70.4) 139 (60.2) Additiveb 0.721 (0.526–0.988) 0.048 0.336 CA 58 (24.9) 79 (34.2) Dominantb 0.636 (0.432–0.935) 0.025 0.175 AA 11 (4.7) 13 (5.6) Recessiveb 0.831 (0.364–1.895) 0.662 1 5-HTTLPR L 107 (23.0) 130 (28.1) Allelea 0.761 (0.566–1.024) 0.071 0.497 S 359 (77.0) 332 (71.9) LL 22 (9.4) 20 (8.7) Additiveb 0.785 (0.594–1.039) 0.100 0.700 LS 63 (27.0) 90 (39.0) Dominantb 0.632 (0.436–0.916) 0.021 0.147 SS 148 (63.5) 121 (52.3) Recessiveb 1.100 (0.583–2.076) 0.842 1 STin2 10 44 (9.4) 43 (9.3) Allelea 1.016 (0.653–1.580) 0.944 1 12 422 (90.6) 419 (90.7) 10/10 5 (2.1) 2 (0.9) Additiveb 1.015 (0.663–1.554) 0.956 1 12/10 34 (14.6) 39 (16.9) Dominantb 0.932 (0.575–1.508) 0.711 1 12/12 194 (83.3) 190 (82.2) Recessiveb 2.511 (0.482–13.080) 0.393 1 HTRA1 rs6295 G 359 (77.0) 358 (77.5) Allelea 1.026 (0.755–1.395) 0.870 1 C 107 (23.0) 104 (22.5) GG 138 (59.2) 138 (59.7) Additiveb 1.026 (0.754–1.398) 0.554 1 GC 83 (35.6) 82 (35.5) Dominantb 1.022 (0.705–1.480) 0.560 1 CC 12 (5.2) 11 (4.8) Recessiveb 1.086 (0.469–2.513) 0.772 1 HTR2A rs6313 G 163 (35.0) 179 (38.7) Allelea 0.868 (0.651–1.111) 0.234 1 A 303 (65.0) 283 (61.3) AA 106 (45.5) 86 (37.2) Additiveb 0.859 (0.663–1.113) 0.252 1 GA 91 (39.1) 111 (48.1) Dominantb 0.711 (0.490–1.030) 0.061 0.427 GG 36 (15.4) 34 (14.7) Recessiveb 1.059 (0.637–1.761) 0.749 1 COMT rs4680 A 143 (30.7) 118 (25.5) Allelea 1.291 (0.969–1.720) 0.081 0.567 G 323 (69.3) 344 (74.5) AA 24 (10.3) 15 (6.5) Additiveb 1.284 (0.65–1.707) 0.118 0.826 GA 95 (40.8) 88 (38.1) Dominantb 1.297 (0.901–1.869) 0.175 1 GG 114 (48.9) 128 (55.4) Recessiveb 1.654 (0.844–3.240) 0.236 1 “A” represent wild type and “a” represent mutant type: allele, a vs. A; additive, aa vs. Aa vs. AA; dominant, aa + Aa vs. AA, recessive, aa vs. Aa +AA. a Chi-square test, b Logistic regression analyses by adjustment for age, gender, educational level and resident location. Pcorr: adjusted by boferroni multiple comparison correction

However, we did not find any significant association SLC6A4 is a putative risk factor for PD and other behav- evidence within the 5-HTTLPR, rs3813034 and STin2 ioral disorders that involve dysregulation of serotonergic polymorphisms of the SLC6A4 gene, which is consistent neurotransmission [45]. Additionally, we found no sig- with findings from other studies and meta-analysis dem- nificant statistical differences in the genotype distribu- onstrating that the 5-HTTLPR and STin2 were not asso- tions or allele frequencies of the SNPs (5-HTR1A ciated with PD [43, 44]. However, inconsistent with rs6295, 5-HTR2A rs6313 and COMT rs4680) between previous research which has found the rs3813034 of PD and control groups. In a study comprised of 119 PD Zou et al. BMC Psychiatry (2020) 20:388 Page 6 of 9

Table 3 Genotype and allele frequencies of serotonin-related gene polymorphisms between responder and nonresponder Gene SNP Alleles and Genotypes Responder Nonresponder Model OR (95% CI) p-value p-valuecorr (n, %) (n = 98) (n = 135) SLC6A4 rs140701 C 37 (18.9) 39 (14.4) Allelea 0.726 (0.443–1.188) 0.201 1 T 159 (81.1) 231 (85.6) CC 5 (5.1) 5 (3.7) Additiveb 0.638 (0.470–1.198) 0.159 1 CT 27 (27.6) 29 (21.5) Dominantb 0.694 (0.391–1.232) 0.160 1 TT 66 (67.3) 101 (74.8) Recessiveb 0.715 (0.201–2.542) 0.446 1 rs3813034 C 155 (79.1) 231 (85.6) Allelea 0.638 (0.394–1.035) 0.067 0.469 A 41 (20.9) 39 (14.4) CC 63 (64.3) 101 (74.8) Additiveb 0.669 (0.423–1.060) 0.113 0.791 CA 29 (29.6) 29 (21.5) Dominantb 0.606 (0.344–1.069) 0.128 0.896 AA 6 (6.1) 5 (3.7) Recessiveb 0.590 (0.175–1.991) 0.332 1 5-HTTLPR L 76 (38.8) 31 (11.5) Allelea 0.205 (0.128–0.328) 0.000 0.000 S 120 (61.2) 239 (88.5) LL 16 (16.3) 6 (4.4) Additiveb 0.249 (0.155–0.401) 0.000 0.000 LS 44 (44.9) 19 (14.1) Dominantb 0.144 (0.079–0.261) 0.000 0.000 SS 38 (38.8) 110 (81.5) Recessiveb 0.238 (0.090–0.634) 0.004 0.029 STin2 10 22 (11.2) 22 (8.1) Allelea 0.702 (0.377–1.307 0.262 1 12 174 (88.8) 248 (91.9) 10/10 3 (3.1) 2 (1.5) Additiveb 0.734 (0.410–1.315) 0.372 1 12/10 16 (16.3) 18 (13.3) Dominantb 0.723 (0.363–1.442) 0.454 1 12/12 79 (80.6) 115 (85.2) Recessiveb 0.476 (0.078–2.905) 0.428 1 HTRA1 rs6295 G 151 (77.0) 208 (77.0) Allelea 1.000 (0.646–1.549) 0.999 1 C 45 (23.0) 62 (23.0) GG 59 (60.2) 79 (58.5) Additiveb 1.000 (0.645–1.551) 0.985 1 GC 33 (33.7) 50 (37.0) Dominantb 1.072 (0.631–1.822) 0.903 1 CC 6 (6.1) 6 (4.4) Recessiveb 0.713 (0.223–2.281) 0.826 1 HTR2A rs6313 G 62 (31.6) 101 (37.4) Allelea 1.292 (0.875–1.906) 0.197 1 A 134 (68.4) 169 (62.6) AA 48 (49.0) 58 (43.0) Additiveb 1.252 (0.869–1.805) 0.199 1 GA 38 (38.8) 53 (39.3) Dominantb 1.274 (0.756–2.149) 0.233 1 GG 12 (12.2) 24 (17.8) Recessiveb 1.550 (0.733–3.274) 0.355 1 COMT rs4680 A 66 (33.7) 77 (28.5) Allelea 0.786 (0.528–1.169) 0.234 1 G 130 (66.3) 193 (71.5) AA 11 (11.2) 13 (9.6) Additiveb 0.793 (0.537–1.172) 0.294 1 GA 44 (44.9) 51 (37.8) Dominantb 0.705 (0.418–1.189) 0.174 1 GG 43 (43.9) 71 (52.6) Recessiveb 0.843 (0.361–1.969) 0.954 1 “A” represent wild type and “a” represent mutant type: allele, a vs. A; additive, aa vs. Aa vs. AA; dominant, aa + Aa vs. AA, recessive, aa vs. Aa +AA. a Chi-square test, b Logistic regression analyses by adjustment for age, gender, educational level and resident location. Pcorr: adjusted by boferroni multiple comparison correction patients and 119 healthy controls from Japanese popula- COMT Val158Met polymorphism has been found to be tion, no significant differences were found in the allele associated with PD in European ancestry, but not Asian frequencies or genotype distributions of the COMT ancestry [46]. Previous studies also found pure PD was rs4680 or the 5-HT1A (rs6295) between PD patients and associated with HTR2A 102 T-C (rs6313) polymorphism controls [10], but other studies have found significant re- [47], but not in other study [14]. The above findings of lationship between the COMT Val158Met (rs4680) poly- genetic association studies are limited and inconsistent. morphism and PD [14, 15]. In a meta analysis, the These mixed results of studies might be attributable to Zou et al. BMC Psychiatry (2020) 20:388 Page 7 of 9

different samples, sex, races, agoraphobia co-morbidity did not find the relationship between other gene poly- or severity of PD. For example, previous study showed morphisms and treatment response to sertraline. This is that the HTR2A polymorphism was associated inconsistent with previous studies that found the 5- with the severity of PD [43]. Above all, these conflicting HT1A receptor -1019C/G polymorphism was strongly findings indicate that the contribution of genetic factors associated with response to treatment in PD receiving to PD may involve a complex network of variants. sertraline or paroxetinethe [54, 59]. Another study sug- In the present study, we only found significant correl- gested that the genetic variant of the COMT enzyme ation between 5-HTTLPR polymorphism and treatment may be related to treatment response to paroxetine in response to sertraline after 4-weeks treatment for PD. PD [60]. Inconsistent results may be related to small The results of our study indicated the PD patients with sample size, short follow-up periods, definition of re- S-allele was linked to poorer response to sertraline dur- sponse, antidepressant choice and ethnic differences. In ing the early stage of treatment, suggesting that 5-HTTL addition, gene-environment interactions may also con- PR could be a predictor of response to sertraline treat- tribute to these inconsistent findings and provide more ment. 5-HTTLPR is a 44 base pair insertion–deletion robust predictors of treatment response. polymorphism which can exist as a long (L) variant of a 16 repeat sequence or a short (S) variant of 14 repeats Conclusions [48]. Previous researches indicated the L allele was asso- The present study suggests that the SLC6A4 rs140701 ciated with higher levels of transcription and concentra- variant may be associated with susceptibility to PD, and 5- tions of 5-HTT mRNA compared to the S allele, and the HTTLPR polymorphism may be a predictor of response short (S) allele of the 5-HTTLPR polymorphism resulted to sertralines in the treatment of PD. However, the results – in less efficient transcription [49 51]. This may explain of our study should be considered preliminary in light of the fact to some extent that the SSRIs are effective in several limitations: (1) The sample size was limited and the treatment of PD with the SLC6A4 being the primary only from Sichuan provinces of western China, which may drug target. These findings may contribute to our under- not completely represent the Chinese ethnicity. (2) The standing of the association between 5-HTTLPR poly- etiology of PD is complex and comprises environmental morphism and antidepressant response. Consistent and genetic factors, a potential gene-environment inter- study found the 5-HTTLPR low-expression genotypes action or epigenetics study should be investigated. (3) We showed a more favorable response to exposure-based be- did not measure the relationship between plasma sertra- havior therapy in PD with agoraphobia [52]. Similar line concentration and clinical response. findings were found in subjects with depressive dis- order treated with sertraline [53]. However, previous re- Supplementary information port found no association of the 5-HTTLPR Supplementary information accompanies this paper at https://doi.org/10. polymorphism with treatment response in a sample size 1186/s12888-020-02790-y. of 102 PD patients treated with sertraline or paroxetine [54]. Also, there was no significant associations between Additional file 1: Table S1. Characteristics of circadian genes and SNPs the 5-HTTLPR polymorphism and sertraline responses in this study in MDD patients [55]. On the one hand, other polymor- Additional file 2: Table S2. The primer sequence in PCR mixture phisms of SLC6A4 may have a combined effect with 5- Additional file 3: Table S3. the probe sequence in probe mixture HTTLPR. In addtion, the variants of cytochrome P450 Additional file 4: Table S4. Power of the study at current sample size enzyme genes may also contribute to the pharmacokin- Additional file 5: Figure S1. PD patients recruit flow chart. etics. One study found the CYP2C19 genetic polymorph- ism is associated with Escitalopram treatment response Abbreviations COMT: Catechol-O-Methyltransferase; CIs: Confidence Intervals; in Chinese patients with PD [56]. On the other hand, CYP450: Cytochrome P450; DSM-IV: Diagnostic and Statistical Manual of functional variants in SLC6A4 may be related to epigen- Mental Disorders fourth edition; HWE: Hardy-Weinberg Equilibrium; L: Long; etic mechanisms and affect gene expression. For in- LD: linkage disequilibrium; MDD: Major Depressive Disorder; ORs: Odds Ratios; PD: Panic disorder; PDSS: Panic Disorder Severity Scale; stance, epigenetic modifications of SLC6A4 gene is PCR: Polymerase Chain Reaction; SCID: Structured Clinical Interview for DSM- showing promising results as biomarkers for prediction IV; SNPs: Single Nucleotide Polymorphism; S: Short; SLC6A4: Solute Carrier of antidepressant response [57]. Notably, evidence has Family 6 Member 4; SSRIs: Selective Serotonin-Reuptake Inhibitors; STin2: intron 2; 5-HTR1A: 1A receptor; 5-HTR2A: 5-Hydroxytryptamine 2A been shown that different ethnicities responded differ- receptor; 5-HTT: Serotonin Transporter; 5-HTTLPR: Serotonin-Transporter- ently to antidepressants possibly due to the variants in Linked Polymorphic Region the SLC6A4 gene. One Meta-analysis showed 5-HTTL PR may be a predictor of antidepressant response and Acknowledgements The authors would like to thank all participants in this study. we thank remission in Caucasians, while it does not appear to play Shanghai Genesky Bio-Tech Co., Ltd. for their excellent technical assistance a major role in Asians [58]. Last but not the least, we with the genotyping analyses. Zou et al. BMC Psychiatry (2020) 20:388 Page 8 of 9

Authors’ contributions 14. Karacetin G, Bayoglu B, Cengiz M, Demira T, Kocabasoglu N, Uysal O, et al. ZLZ was critically involved in the study design and wrote the manuscript. Serotonin-2A receptor and catechol-O-methyltransferase polymorphisms in YLH, JYW and WJM were involved in subject recruitment. BZ guided this panic disorder. Prog Neuro-Psychopharmacol Biol Psychiatry. 2012;36:5–10. research and supervised the entire project. All authors read and approved 15. RotheC,KoszyckiD,BradwejnJ,KingN,DeLuca,V,TharmalingamS,etal. the final manuscript. Association of the Val158Met catechol O-methyltransferase genetic polymorphism with panic disorder. Neuropsychopharmacology. 2006; 31:2237–2242. Funding 16. Annerbrink K, Westberg L, Olsson M, Olsson M, Allgulander S, Andersch S, et al. This study was supported by grants from National Natural Science Association between the catechol-O-methyltransferase Val158Met polymorphism Foundation of China(81801360). In addition, this study was also supported and panic disorder: a replication. Psychiatry Res. 2010;178:196–8. by grants from the Chinese academy of medical sciences (2019PT310020). 17. Kim H, Lim SW, Kim S, Kim JW, Chang YH, Carroll BJ, et al. Monoamine The funding bodies played no role in the design of the study and collection, transporter gene polymorphisms and antidepressant response in koreans analysis, and interpretation of data and in writing the manuscript. with late-life depression. JAMA. 2006;296:1609–18. 18. Dong ZQ, Li XR, He L, He G, Yu T, Sun XL. 5-HTR1A and 5-HTR2A genetic Availability of data and materials polymorphisms and SSRI antidepressant response in depressive Chinese The datasets used and analyzed during the current study are available from patients. Neuropsychiatr Dis Treat. 2016;12:1623–9. the corresponding author on reasonable request. 19. Myung W, Lim SW, Kim S, Kim H, Chung JW, Seo MY, et al. Serotonin transporter genotype and function in relation to antidepressant response in Ethics approval and consent to participate Koreans. Psychopharmacology. 2013;225:283–90. The study was approved by the Sichuan Provincial People’s Hospital ethics 20. Corregiari FM, Bernik M, Cordeiro Q, Vallada H. and committee, reference number: (2016) Ethics Review (29). All individuals serotonergic polymorphisms associated with treatment response in provided written informed consent prior to the initiation of study obsessive-compulsive disorder. Clinics (Sao Paulo). 2012;67:335–40. procedures. 21. Kato M, Fukuda T, Wakeno M, Okugawa G, Takekita Y, Watanabe S, et al. Effect of 5-HT1A gene polymorphisms on antidepressant response in major depressive Consent for publication disorder. Am J Med Genet B Neuropsychiatr Genet. 2009;150B:115–23. Not applicable. 22. Gudayol-Ferré E, Herrera-Guzmán I, Camarena B, Cortés-Penagos C, Herrera- Abarca JE, Martínez-Medina P, et al. 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